Characterization of reaction variables

ABSTRACT

A microscale method for the characterization of one or more reaction variables that influence the formation or dissociation of an affinity complex comprising a ligand and a binder, which have mutual affinity for each other. The method is characterized in comprising the steps of: (i) providing a microfluidic device comprising a microchannel structures that are under a common flow control, each microchannel structure comprising a reaction microactivity; (ii) performing essentially in parallel an experiment in each of two or more of the plurality of microchannel structures, the experiment in these two or more microchannel structures comprising either a) formation of an immobilized form of the complex and retaining under flow conditions said form within the reaction microactivity, or b) dissociating, preferably under flow condition, an immobilized form of the complex which has been included in the microfluidic device provided in step (i), at least one reaction variable varies or is uncharacterized for said two or more microchannel structures while the remaining reaction variables are kept essentially constant; (iii) measuring the presentation of the complex in said reaction microactivity in said two or more microchannel structures; and (iv) characterizing said one or more reaction variables based on the values for presentation obtained in step (iii).

TECHNICAL FIELD

The present invention relates to a microscale method for thecharacterization of at least one reaction variable that influence theformation or dissociation of an affinity complex comprising a ligand anda binder. The ligand and the binder have affinity to each other and areaffinity counterparts.

BACKGROUND TECHNOLOGY

Microscale methods have previously been used for finding newcombinations of ligands and binders for screening libraries of compoundsfor

(a) new drug candidates which are based on a receptor-ligandinteraction, and

(b) new ligand candidates for affinity assays, affinity chromatographyetc.

Microscale methods have also been suggested for optimizing theconditions for formation or dissociation of a particular affinitycomplex or when selecting affinity counterparts for other reasons.

Microscale methods have also been used for determining the amount of aparticular compound (analyte) in a sample.

Methods have been used that are based on affinity capture of a binder oran analyte by an affinity counterpart that is immobilized orimmobilizable to a solid phase.

Typically the formation of the complex on a solid phase has beenperformed under non-flow-conditions. See for instance Eteshola et al,Sensors and Actuators B 72 (2001) 129-133; Sato et al, Anal. Chem. 72(2000) 1144-1147; and WO 9721090 (Gamera, Mian et al).

Non-flow conditions will simplify the methods but information that onlycan be obtained from experiments performed under flow conditions will bemissed. For small molecules non-flow conditions may be acceptablebecause their diffusion rates are often relatively high and orientationof their binding sites for binding is uncomplicated. This is normallynot applicable to larger molecules. Without controlling the liquid flowaccurate information will be difficult to obtain for large molecules.

Recently a MALDI MS integrated microfluidic affinity system based onaffinity binding to a reverse phase matrix has been presented. In thissystem a protein digest was adsorbed to a reverse phase matrix andsubsequently desorbed and transported to an outlet port that functionedas a MALDI MS target. (Integrated sample preparation and MALDI MS on amicrofluidic compact disc (CD with improved sensitivity) (MagnusGustavsson et al) ASMS 2001. The demand on the reproducibility in thebinding, the control of the liquid flow rate, and the residence time waslow.

WO 0138865 (Univ. of Alberta, Harrison et al) describes a solid phaseextraction method in a singular microchannel structure utilizing flowconditions and affinity binding. In another example the publicationshows saturation of solid phase bound anti-theopyhilline antibodies withtheophylline.

DEFINITIONS

Reaction variables are mainly of two kinds: 1) variables related toaffinity reactants with the subgroups a) amounts including presenceand/or absence, and b) properties of affinity reactants includingaffinity, and 2) reaction conditions.

Group 1a comprises amounts in mass units, molar units, relative amounts,concentrations, activity/mass unit, activity/volume unit, relativeactivities etc. Activity refers to (i) biological activity, (ii) bindingactivity for an affinity counterpart etc. Biological activity includesenzyme activity binding activity towards a native or synthetic affinitycounterpart, cell activity etc.

Group 1b comprises properties of an affinity reactant (a ligand and/or abinder).

Typically this group comprises properties of inhibitors and promotorsfor the formation or dissociation of an affinity complex, enzymes,substrates, cofactors, cosubstrates, coenzymes, receptors, hormones,antibodies and antigen/hapten-binding fragments thereof,antigens/haptens etc. Synthetic and recombinant forms are included.

If a variable of group 1b varies or are different between samples,microchannel structures, reaction microcavities, aliquots of liquid etcthis primarily means that different molecular entities are present inrespective unit.

The properties related to group 1b comprises affinity which includesaffinity constants (and the corresponding constant for thedissociation), rates for formation and dissociation, knowledge aboutcounterpart(s) towards which a binder has/have affinity and vice versa(specificity, selectivity etc) etc. Affinity also comprises relativeaffinity of different complexes, for instance a common ligand versus arange of binders and vice versa, a common complex versus changes inreaction conditions etc.

Reaction conditions in this context means reaction variables that arenot related to a property of an affinity reactant. Reaction conditionscomprise pH, temperature, ionic strength (including type of salts),hydrogen bond breaking agents (denaturing agents, amount and kind)),detergents (amount and kind), liquid flow, immobilization techniques,solid phases etc.

Affinity reactants (ligands and binders) that are to be characterizedwith respect to group 1 variables are called “analytes”. The term“Analyte” also includes analyte-derived entities that originate from anoriginal analyte in an original sample that has been processed to thesample used in the microfluidic device for step (ii). This preprocessingmay take place outside the microfluidic device and/or in separatesubstructures within the microfluidic device. The amount of ananalyte-derived entity in a processed sample is a function of theoccurrence of the original analyte in the original sample.

The term “analogues” is used for two or more binders or ligands that arecapable of inhibiting or competing with each other for affinity bindingto a common affinity counterpart. In the same manner “analogue” is alsoused for an analyte.

A microfluidic device comprises at least one microchannel structurethrough which liquid flow is used for the transport of reactants

The terms “microformat”, “microchannel” etc contemplate that amicrochannel structure comprises one or more cavities and/or channelsthat have a depth and/or a width that is ≦10³ μm, preferably ≦10² μm.The volumes of microcavities/microchambers are typically ≦1000 nl(=nl-range), such as ≦500 nl or ≦5100 nl or ≦50 nl, but may also belarger, i.e. in the interval 1-1000 μl, such as 1-100 μl or 1-10 μl.

The term “microfluidic method” means that (a) the method is performed ina microfluidic device, and (b) the liquid volumes are in themicroformat.

A “library” is a set of two, three, four or more compounds orsubstances, for instance ≧10, such as ≧100 or ≧1000 or ≧10000. Typicalexamples are recombinant, synthetic or native sets of nucleotides,oligonucleotides, polynucleotides, amino acids, oligopeptides,polypeptides, proteins, lipids, other organic and inorganic compounds,carbohydrates, bacteria, phages, polymers, biopolymers etc. Eachindividual library may contain members of different classes, forinstance polypeptides and polynucleotides, carbohydrates and proteinsetc. A library may also include sets that are derived from nature, e.g.sets of native proteins in which the individual proteins relates to eachother due to post-translational modifications, isoproteins such asisoenzymes etc. Examples of post-translational modifications that canresult in a library are phosphorylation, activating cleavage,glycosylation, ubiquitination, etc.

The term “library” also includes sets of other reaction variables, suchas different pH values, different immobilization groups, different ionicstrengths, different concentrations, different salts, different solidphases, different spacers etc.

The “library” also includes a collection of samples (aliquots of liquid)differing with respect to at least one reaction variables.

OBJECTS OF THE INVENTION

A first object is to provide an improved microscale method forquantifying the amount of an analyte in a plurality of samples by usingassay methods that are based on specific affinity binding and capture toa solid phase

A second objective is to provide a fluidic function that whenincorporated into the microchannel structures of a microfluidic devicewill standardize the flow rate in the individual microchannel structuresin a microfluidic device, i.e. to control the flow rate such that theinter-channel variation is reduced to an acceptable level.

A third objective of the present invention is to provide a robustmicrofluidic system and method that can be used for parallel screeningof reaction variables in order to find new and optimal binder-ligandcombinations and/or to grade affinity for a range of affinity complexes,ligands and binders, and/or to optimize processes involving formation ordissociation of immobilized affinity complexes under flow conditions.

These objects primarily concern methods which utilize interactionsinvolving at least one affinity reactant which is relatively largeand/or comprises one or more functional groups comprising a heteroatomselected amongst nitrogen, oxygen, sulphur and phosphorous. Relativelylarge in this context means that the affinity reactant simultaneouslycan bind two affinity counterparts and/or has a molecular weight ≧1000dalton, such as ≧2000 or ≧10000 dalton. Typically one of the affinityreactants is polymeric, such as a biopolymer comprising carbohydratestructure and/or peptide structure and/or nucleotide structure and/orlipid structure. Molecules that are not large are considered to besmall.

FIGURES

FIGS. 1 a and 1 b illustrate a variant of a preferred microchannelstructure that has a narrow microconduit that create a significantpressure drop. This variant has been used for the model study presentedin the experimental part. FIGS. 1 a and 1 b are identical except thatFIG. 1 a gives the dimensions and FIG. 1 b hydrophobic surface breaksand their dimensions.

FIG. 2 illustrates a set of microchannel structures of the same kind asused in the experimental part. The structures are linked together by acommon distribution channel and a common waste channel.

FIG. 3 illustrates the key part of a substructure that comprise areaction microcavity and means for creating a pressure drop.

FIG. 4 represents the results obtained in the experimental part.

The structures shown in FIGS. 1-2 are intended to be placed on acircular disc around its axis of symmetry (spinning axis). The arrowshows the direction towards the center of the disc. Note also thearc-like configuration.

SUMMARY OF THE INVENTION

The present inventors have recognized that the method defined under“Technical Field” above can be improved by

-   (a) utilizing a microfluidic device comprising two or more    microchannel structures in which the flow is under a common flow    control, and-   (b) performing formation of the affinity complex under flow    conditions in at least two of the microchannel structures and    retaining an immobilized form of the formed affinity complex under    flow conditions in a reaction microcavity in each of the    microchannel structures used.

In a similar manner the principle of common flow control and flowconditions are applied to the dissociation of an affinity complexretained in the reaction microcavity for the characterization ofreaction variables influencing the dissociation reaction.

The inventors have also recognized a number of additional features thatcan reduce the effects of variations in flow between differentmicrochannel structures within the same device (inter-channelvariation):

-   (a) A means for flow restriction downstream the reaction microcavity    in each microchannel structure for creating a significant pressure    drop.-   (b) A porous matrix placed in a desired part, for instance the    reaction microcavity or immediately downstream the reaction    microcavity, of each microchannel structure for creating a    significant pressure drop.-   (c) A packed bed of monosized particles instead of polysized    particles in the reaction microcavity.-   (d) a pulse giving increased flow will assist in over-coming    inter-channel variations in flow resistance, in particular when    initiating flow and/or when the liquid is to pass through branchings    and curvatures.-   (e) Anti-wicking means in inner edges downstream and in close    proximity to the outlet end of the outlet microconduit which leads    waste liquids from a reaction microcavity.-   (f) Excess of solid phase affinity reactant (e.g. binder    (immobilized reactant)) in the reaction microcavity.-   (g) Selecting flow rates that accomplish residence times ≧0.010    seconds for the formation of complex in the reaction microcavity.

Significant pressure drop is relative to inter-channel variations inflow resistance.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a microscale method for characterizing at least onereaction variable. The method is generally defined in the firstparagraph. The characterizing feature comprises four steps:

-   Step (i): Providing a microfluidic device comprising a plurality of    microchannel structures that are under common flow control. Each of    the structures comprises a reaction microcavity for retaining an    affinity complex.-   Step (ii): Performing essentially in parallel an experiment in each    of two or more of the plurality of microchannel structures. Each    experiment comprises:    -   (a) forming an immobilized form of the complex and retaining        under flow conditions this form within the reaction microcavity,        or    -   (b) dissociating an immobilized form of the complex within the        reaction microcavity.    -   The reaction variables referred to are different and/or are        uncharacterized for two or more of the experiments. Other        reaction variables are kept essentially constant. Dissociating        is typically performed under flow conditions. The immobilized        form in (b) is introduced prior to step (ii). The formation of        the retained complex is a function of the introduction of the        ligand and the binder.-   Step (iii): Measuring the presentation of the complex in the    reaction microcavity in each of said two or more microchannel    structures.-   Step (iv): Characterizing said at least one reaction variable based    on the values for presentation which is measured in step (iii).

The experiments are initiated by introducing the appropriate liquidaliquots into inlet ports of the device and applying the appropriatedriving force for liquid flow.

In the same microfluidic device there may also be run other similar ordissimilar experiments in parallel with those defined above.

The term “essentially constant” in this context means that the othervariables are kept within ranges where the outcome of the experimentswill be essentially the same.

In the preferred variants, the reaction microcavities to be used in themethod and provided in step (i) comprise an affinity reactant (affinitybinder) that in step (ii) will be incorporated into the immobilizedcomplex.

Common Flow Control and Liquid Flow Conditions for Step (Steps (i) and(ii)).

The term “common flow control” means that when a driving force for aliquid flow is applied in one part of a microchannel structure(structure 1), there will also be applied a driving force for liquidflow in the corresponding part of each of the other microchannelstructures of the device. The driving force in the individual channelsderives from the same source, e.g. spinning the device if centrifugalforce is the driving force. Moreover, an increase or a decrease indriving force in one microchannel structure is paralleled with anincrease or a decrease in the other microchannel structures. The size ofthe force (and the liquid flow rate) may differ between differentmicrochannel structures for which common flow control is applied. Incentrifugal based systems, for instance, the designs of the microchannelstructures may differ and/or the microchannel structures may be placedat different radial distances.

Common flow control primarily refers to the flow through the reactionmicrocavity (solid phase) when the immobilized complex is formed andretained. Flow control may be less critical in other parts of thestructures and/or for other steps of the method.

The liquid flow may be driven by distinct means that either is presentin or external to the device. Thus, liquid flow may be created byelectroosmosis, micropumps, expanding gas etc. Another alternative is touse forces such as capillary force and inertia force includinggravitational force and centrifugal force to drive the liquid, i.e.forces which do not require any means on the microfluidic device.

The appropriate flow rate through the reaction microcavity (step (ii))depends on a number of factors, such as the affinity pair used forforming the immobilized complex, the volume of the reaction microcavity,the solid phase in the microcavity etc. Typically the flow rate shouldgive a residence time of ≧0.010 seconds such as ≧0.050 sec or ≧0.1 secwith an upper limit that typically is below 2 hours such as below 1hour. Illustrative flow rates are within 0.01-100 nl/sec, typically0.1-10 nl/sec. Residence time refers to the time it takes for a liquidaliquot to pass through the reaction microcavity (i.e. through the solidphase).

An acceptable flow control depends on a particular application includingconcentrations of reactants, their diffusion properties and reactionrates, etc. Sufficient flow control in most cases means that theintra-channel variation for residence time is within the mean residencetime ±90%, such as ±75% or ±50% or ±25%.

According to a preferred variant of the invention, common flow controlis accomplished by spinning a microfluidic device in which themicrochannel structures are oriented from an inward position to anoutward position in relation to an axis of symmetry of a substrate andutilizing the centrifugal force for driving the liquid. Common flowcontrol also include that centrifugal force is used to create asufficient local hydrostatic pressure within a structure to drive aliquid aliquot through an outward (downward) and/or an inward (upward)bent of a microchannel structure. See for instance WO 0146465 (GyrosAB).

Typical spinning speeds are within the interval 50-25000 rpm, such as50-15000 rpm. The spinning speed within a given protocol may vary, forinstance comprise sequences with individual ramps of acceleration,deceleration, and constant spinning. It may be beneficial to include apulse of increased spinning at certain positions. See above.

Microfluidic Device (Step (i)).

The present invention is primarily intended for geometric arrangementsin which the microchannel structures are present in a substrate(microfluidic device) that has an axis of symmetry. Each microchannelstructure is then oriented in an outward direction relative to the axisof symmetry, i.e.

-   (a) the reaction microcavity is part of a substructure which also    comprises a structural unit delivering liquid to the reaction    microcavity, and-   (b) the latter unit is at a shorter radial distance than the    reaction microcavity and communicates with an inlet port.

An inlet port is typically located at a shorter radial distance than thereaction microcavity. Downstream the reaction microcavity there may bean outlet port, typically located at a larger radial distance than thereaction microcavity. By utilizing capillary force and/or othernon-centrifugal forces for the introduction of a liquid into amicrochannel structure, inlet ports may be located at in principle anyradial distance, e.g. more remote from the axis of symmetry than anoutlet port and/or the reaction microcavity.

The microchannel structures may or may not be oriented in a planeperpendicular to the axis of symmetry.

In centrifugal based systems a “higher” or an “upper” level/positionwill be at a shorter radial distance (inner position) compared to a“lower” level/position (outer position). Similarly, the terms “up”,“upward”, “inwards”, and “down”, “downwards”, “outwards” etc will meantowards and from, respectively, the spinning axis. This applies if nototherwise is specified. With respect to other arrangement/substrates andconventional driving forces, i.e. gravity force, externally appliedpressure, electro-osmotically driven flows etc, these terms have theirconventional meaning.

Axes of symmetry are n-numbered (C_(n)) where n is an integer between 2and ∞, preferably 6, 7, 8 and larger, for instance ∞. In preferred casesthe substrate (microfluidic device) as such may have a circular,cylindrical, spherical or conical symmetry (C_(∞)).

The preferred devices are typically disc-shaped with sizes and formssimilar to the conventional CD-format, e.g. in the interval from 10% upto 300% of the conventional CD-radii.

Suitable microfluidic devices may be manufactured from a planarsubstrate surface comprising a plurality of uncovered microchannelstructures that in a subsequent step are covered by another planarsubstrate (lid). See WO 9116966 (Pharmacia Biotech AB) and WO 0154810(Gyros AB). At least one of the substrates may be transparent, e.g. thesecond substrate (lid). Both substrates are preferably fabricated fromplastic material, e.g. plastic polymeric material.

Different applications require different surface characteristics. Thismeans that in the case aqueous liquids are to be transported within themicrochannel structures hydrophilization may be required. See forinstance WO 0056808 (Gyros AB) and WO 0147637 (Gyros AB). For aqueousliquids an essential part of the inner surfaces should have watercontact angles ≦90°, such as ≦40° or ≦30° or ≦20° at the temperature ofuse. At least two or three of the inner walls enclosing the channelsshould comply with this range. Surfaces in passive valves, anti-wickingmeans etc are excluded from these general rules.

Non-wettable surface breaks may be introduced at predetermined positionsin the inner walls of the microchannel structures before covering theuncovered microchannel structures (WO 9958245, Amersham PharmaciaBiotech AB) and WO 0185602, Amic AB & Gyros AB). For aqueous liquidsthis means hydrophobic surface breaks. Surface breaks may be used forcontrolling the liquid flow within the structures, e.g. anti-wicking,passive valves, directing liquids etc. See below. In FIG. 1 a, surfacebreaks are cross-hatched and openings (inlets, outlets, vents) in thecovering substrate (lid) are represented by hatched circles.

FIGS. 1 a-b and 2 illustrate a microchannel structure (101) that can beused in the present invention. The structure comprises a reactionmicrocavity (104) connected to one or more inlet microconduits (102 and103) and an outlet microconduit (105) with an outlet end (106). When thestructure is used the immobilized complex becomes retained in thereaction microcavity (104). Alternatively the reaction microcavitycontains the affinity complex from the beginning and the method meansdissociation of the complex.

If there are two or more inlet microconduits (102 and 103) theytypically merge before reaching the reaction microcavity (104). In thecase the reaction microcavity contains a solid phase in form ofparticles the trespass into the outlet microconduit (105) typically is asharp drop in the cross-sectional area that prevents the particles frompassing into the outlet microconduit.

For centrifugal based systems, two inlet microconduits (102 and 103)typically form at downward bent (107) with shanks corresponding to theinlet microconduits (102 and 103). The outlet microconduit (105) isconnected to the lower part of the bent via the reaction microcavity(104).

An inlet microconduit (102,103) may be connected to an inlet port(108,208 and 209 and 210) via a functional unit. This unit may be avolume-defining unit (111) that comprises a metering microcavity (112)connected to one of the inlet microconduits (102), an overflow channel(113) that starts in a narrow conduit part between the metering cavity(112) and the inlet unit (114) and ends in a waste function, forinstance comprising a common waste reservoir/channel (115,215). Thewaste function may have one or more outlet ports (116216). Thevolume-defining unit (111) comprises valve functions (117,118) that areassociated with the overflow channel (113) and with the outlet end ofthe metering microcavity (112). For centrifugal based systems thesevalve functions are typically passive and preferably based on localchanges in surface characteristics. The valve function (118) is at alarger radial distance than the valve function (117). This means thatthe positions of these valves are selected to support that liquid in theoverflow channel (113) is passed into the waste function at a lowerspinning speed than liquid in the metering microcavity (112) is passedinto the inlet microconduit (102). A volume-defining unit of this kind(111) is primarily intended for liquid aliquots that are to beintroduced with high accuracy with respect to volume. This means liquidaliquots that contain

-   (a) an analyte and/or-   (b) a reaction variables of group 2 that varies between microchannel    structures and/or-   (c) any other reagent that has to be delivered with a high accuracy.

Aliquots of type (a) and (b) will be called aliquot 1.

An alternative functional unit connected to one of the inletmicroconduits (102,103) is a unit for distributing liquid aliquots toseparate microchannel structures. The unit may be in the form of adistribution channel (119,219) that is common for several microchannelstructures (101,201). For centrifugal based system the channel may havealternating upper and lower parts (120,220 and 121,221, respectively)with an inlet vent (122,222) to ambient atmosphere in each upper part(120) and liquid communication in each lower part (121) via a valvefunction (123) to one of the inlet conduits (103,203) of eachmicrochannel structure (101,201). The vents (122,222) communicate withambient atmosphere via a common venting channel (124,224). Thedistribution channel may have one or more inlet ports (209,210) and oneor more outlet ports (125,225) (only one shown).

As shown in the figures the outlet end (106) may mouth into a wasteconduit chamber (126) (belonging to the microchannel structure). Thischamber in turn may mouth into a common waste conduit or a common wastereservoir (115). Although not preferred, the outlet conduit (105) may inan alternative variant mouth directly into a common waste chamber orinto ambient atmosphere. For centrifugal based systems utilizing passivevalves, the outlet end (106) of the outlet microconduit (105) typicallyis at the same or a higher level than the reaction micocavity(preferably its top part) (104). According to one inventive aspect ofthe invention the outlet microconduit (105) possibly in combination witha solid phase in the reaction microcavity (104) are able to produce apressure drop against a liquid flow passing through the microchannelstructure, which is significantly larger that the resistance to flowupstream the reaction microcavity (104). The pressure drop in amicroconduit is proportional to its length and inversely proportional toits hydrodynamic cross-sectional area.

In the invention the pressure drop across the outlet microconduit (105)between the reaction microcavity (104) and the waste chamber (126)should be large enough to level out intra-channel differences(variations) in flow resistances that primarily depend on (a) innersurface characteristics upstream the reaction microcavity and (b) thesolid phase used (for instance the packing geometry may differ).

The principles outlined have lead to the design of the microchannelstructures used in the experimental part (FIGS. 1-2):

-   (a) Reaction microcavities (104) with a depth of 100 μm and width of    250 μm,-   (b) Outlet microconduits (105) with a depth of 10 μm, a width of 20    μm and a length of 4.56 mm.

Other parts of the microchannel structures had a depth of 100 μm.

There may be vents to ambient atmosphere at appropriate positions to

-   (a) level out over-pressure that may be created in “dead-ends” when    liquid is introduced into a microchannel structure, or-   (b) interrupt flow at certain positions.

In the variant illustrated in the FIGS. 1-2, the second kind is presentin inlet port (108,208). Venting functions (122,222 and 127,227) thatbreak a liquid flow are also present in the common distribution channeland in the waste chamber (119,219 and 126,226, respectively).

The inlet ports (108,208 and 209 and 210) preferably are connected to amicrocavity (128), which typically is narrowing inwards the microchannelstructure and has longitudinal projections (ridges) (129) in the flowdirection. These ridges will facilitate quick transport of a dispensedliquid aliquot into the interior of an inlet unit. The inlet ports alsomay have a non-wettable area (typically hydrophobised) (130,cross-hatched) that will direct a dispensed liquid into the inlet unit(114).

In order to avoid undesired loss and transport of liquid via wicking,the microchannel structures may be equipped with anti-wicking means atselected positions in form of changes in surface characteristics. Thechanges are typically local and may be related to geometric surfacecharacteristics (131) and/or chemical surface characteristics (132,133)(cross-hatched area). See WO 9958245, Amersham Pharmacia Biotech AB) andU.S. Ser. No. 60/315,471 and the corresponding International PatentApplication. For aqueous liquids this means that the change is fromhydrophilic to hydrophobic. There is also an anti-wicking function(134,135,123,117) (cross-hatched) present at the inlet vents (127,122)and in the passive valves (123,117).

Valves are preferably passive, i.e. passage of a liquid will depend onthe applied driving force and the physicochemical match between a liquidand the inner surface at the valve position. No movable mechanical partsare needed. Examples are capillary valves that are based purely on achange in geometric surface characteristics (WO 9615576 (David SarnoffRes. Inst.) and WO 9807019 (camera). Preferred passive valves are basedon a change in chemical surface characteristics, i.e. non-wettablesurface breaks. For aqueous liquids this means hydrophobic surfacebreaks (WO 9958245, Amersham Pharmacia Biotech AB). Valves may also bebased on a combination of changes in both chemical and geometric surfacecharacteristics. The changes are typical local. See also U.S. Ser. No.60/315,471 and the corresponding International Patent Application. Otherkinds of valves may also be used.

More details about inlet units, distribution units, volume-definingunits, waste conduits, anti-wicking means and valves, in particular forcentrifugal based systems, are given in U.S. Ser. No. 60/315,417 and thecorresponding International Patent Application.

Reaction Microcavity and Solid Phase.

The geometry of reaction microcavity (104) is preferably a straightmicrochannel that that may be continuously widening and/or narrowing. Atleast a part of the wall of the reaction microcavity is transparent withrespect to the principle used for measuring the presentation of thecomplex.

The reaction microcavity preferably comprises a catching affinityreactant immobilized to a solid phase, which is kept within the reactionmicrocavity.

The solid phase is derivatized with a reactant (capturing reactant) thatcan be incorporated by affinity into the affinity complex that is formedin immobilized form in step (ii). The capturing reactant may be animmobilized form of the ligand or the binder.

The solid phase may be the surface of the inner wall of the microcavity(104), the inner surface of a porous monolith that wholly or partly willoccupy the interior of the reaction microcavity or a population ofporous or non-porous particles that are packed to a bed. In the case thesolid phase comprises particles there should be retaining meansassociated with the downstream end of the reaction microcavity. Thismeans is preferably in the form of a constriction, e.g. in the form of abarrier, that prevents the particles from leaving the microcavity. Theparticle diameter/size should at least be of the same size as or largerthan the smallest dimension of the opening in the constricted part.Another kind of retaining means is magnetic particles combined with anexternally applied magnetic field.

A porous monolith may be fabricated in one piece of material or maycomprise particles that are attached to each other.

By the term “porous particles” is meant that the particles can bepenetrated by soluble reactants that are to be incorporated into theaffinity complex. This typically means Kay values within the interval of0.4-0.95 for these reactants. Non-porous particles have a Kay-valuebelow 0.4 with respect to the same reactants. Porous monoliths havepores that are large enough to permit mass transport of the reactantsthrough the matrix by the liquid flow applied.

The particles may be spherical or non-spherical. With respect tonon-spherical particles, diameters and sizes refer to the “hydrodynamic”diameters.

The particles are preferably monodisperse (monosized) by which is meantthat the population of particles placed in a reaction microcavity has asize distribution with more than 95% of the particles within the rangeof the mean particle size ±5%. Population of particles that are outsidethis range are polydisperse (polysized).

The solid phase may or may not be transparent for the principle used formeasuring the complex.

The material in the solid phase, e.g. the particles, is typicallypolymeric, for instance a synthetic polymer or a biopolymer. The termbiopolymer includes semi-synthetic polymers comprising a polymer chainderived from a native biopolymer. The particles and other forms of solidphases are typically hydrophilic in the case the liquid flow is aqueous.In this context hydrophilic encompasses that a porous solid phase, e.g.a packed bead, will be penetrated by water. The term also indicates thatthe surfaces of the particles shall expose a plurality of polarfunctional groups in which there is a heteroatom selected amongstoxygen, sulphur, and nitrogen. Appropriate functional groups can beselected amongst hydroxy groups, straight eythylene oxide groups([—CH₂CH₂O—]_(n), n an integer >0), amino groups, carboxy groups,sulphone groups etc, with preference for those groups that are neutralindependent of pH. A hydrophobic particle may be hydrophilized, forinstance by introducing hydrophilic groups. See for instance theexperimental part. The coating technique is similar to the techniquepresented in WO 9800709 (Pharmacia Biotech AB, Arvidsson & Ekström).

The techniques for immobilization may be selected amongst those that arecommonly known in the field, for instance the bonding to the solid phasemay be solely via covalent bonds, affinity bonds (for instancebiospecific affinity bonds), physical adsorption (mainly hydrophobicinteraction) etc. Examples of biospecific affinity bonds that can beused are bonds between strepavidin and a biotinylated affinity reactant,high affinity antibody and a haptenylated affinity reactant etc.

Other Functional Units in the Microchannel Structures.

A microchannel structure may also comprise separate or combined unitsthat enables:

-   (1) separation of particulate matter from a liquid aliquot    introduced via an inlet port,-   (2) mixing, e.g. for the formation of an affinity complex upstream    the reaction microcavity, and-   (3) detection of (a) a reactant that has passed through the reaction    microcavity, or (b) a component released from the complex formed in    the reaction microcavity.

Except for unit 3, these functional units if present may be are locatedupstream the reaction microcavity.

A unit for separation of particulate matter is typically positionedupstream a volume metering unit or the two units are combined in acommon unit.

Preferred separation units, volume metering units and mixing units aregiven in U.S. Ser. No. 60/315,417 and the corresponding InternationalApplication.

Additional units may also be present.

The presence of units 1-2 means that the protocol used for step (ii) mayinclude at least one substep selected from

-   -   (ii.a) a separation step, and    -   (ii.b) a mixing step that may include formation of the affinity        complex that is retained in immobilized form in the reaction        microcavity.

The separation step may be carried out simultaneously with avolume-metering step if each of the microchannel structures used canutilize a combined separation/volume metering unit. Otherwise theseparation step is typically upstream a volume-metering step, inparticular if the separation step is used to clean a sample. A mixingstep, if present, is typically downstream a volume metering step.

A separate detection microcavity may enable measuring of:

-   -   an analytically detectable entity that is released from the        complex retained in step (ii),    -   an enzyme product in the case the complex has enzyme activity        and a substrate has been passed through the reaction microcavity        subsequent to step (ii),    -   a compound passing through the reaction microcavity as a        functional control,    -   a second analyte by retaining an immobilized form of a complex        that is different from the complex retained in step (ii),    -   a standard compound (calibrator) that is passing through the        reaction microcavity without interfering with steps (ii) and        (iii).

The first two alternatives may also be part of step (iii).

Protocols Comprising Formation or Dissociation of an Immobilized Complexin the Reaction Microcavity (Step (ii)).

A. Determination of an Uncharacterized Amount of an Analyte.

These protocols are selected amongst biospecific affinity assays thatare used for the determination of an analyte in a sample. The principlesare well known in the field. The protocols encompass that one or moreaffinity counterparts to the analyte are used for the formation of anaffinity complex which is then measured and related to the amount of theanalyte in a sample. The assay conditions are selected such that theamount of the complex becomes a function of the amount of analyte in thesample.

In the variants used in the invention, the complex to be measuredcorresponds to the immobilized complex retained in a reactionmicrocavity in step (ii). Depending on the protocol used this complexmay or may not comprise the original analyte of the original sampleintroduced into the microfluidic device. Reaction variables other thanthe amount of analyte are in principle kept essentially constant forexperiments run in different microchannel structures.

Step (ii) may comprise introduction of one or more additional reactantsthat will become incorporated into the retained complex in order tofacilitate its measurement in step (iii). Similarly step (iii) maycomprise introduction of one or more additional reactants that might beneeded for the measurement of the complex formed in step (ii).

The affinity reactant that defines the link between the solid phase andthe immobilized complex to be measured in step (iii) may be introducedby the manufacturer and therefore provided in step (i). Other reactants,including the analyte, are introduced in subsequent steps. This may bedone in sequence, in parallel, and/or as mixtures. One or moreadditional inlet ports may be used. If needed, mixing of affinityreactants and liquids may take place within separate mixing units thatare located upstream the reaction microcavity.

According to the invention, amounts can be determined during bothdiffusion-limiting and non-diffusion-limiting conditions if proper carehas been taken. The flow rate may in principle be used to secure thatthe conditions contemplated are at hand, the general guide-line beingthat a decrease in flow rate (increase in residence time) will favornon-diffusion limiting conditions and vice versa for diffusion-limitingconditions. These rules primarily apply to large molecules.

Competitive/Inhibition Protocols.

In these protocols the analyte and an analyte analogue are competingwith each other for binding to a limiting amount of an affinitycounterpart to the analyte. This counterpart may be

-   (a) immobilized or immobilizable if the analyte analogue is soluble    and analytically detectable, and-   (b) analytically detectable if the analyte analogue is immobilized    or immobilizable.

At the filing date variant (b) is of great interest for the invention.This variant includes that the analyte and its affinity counterpart arepreincubated before reaching the reaction microcavity, for instanceoutside the microfluidic device or in a separate mixing microcavityupstream the reaction microcavity. The mixture is transported throughthe reaction microcavity where the free (=uncomplexed) affinitycounterpart forms an affinity complex with an immobilized analyteanalogue. This complex is subsequently measured in step (iii).

Competitive variants also include displacement assays in which animmobilized or immobilizable affinity complex comprising two affinitycounterparts is incubated with a sample containing an analyte. Providedthat one of the affinity counterparts is an analytically detectableanalyte analogue, the analyte will displace the analyte analogue in thecomplex, which also means that the signal from the complex will change.

A preferred displacement assay is to provide this kind of complex inimmobilized form in the reaction microcavity in step (i). In step (ii)the analyte will displace the analyte analogue of the immobilizedcomplex. In step (iii) the amount of complex containing the analyte ismeasured directly in the reaction microcavity or indirectly fromreleased analytically detectable analyte analogue in a separatedetection microcavity downstream the reaction microcavity.

Competitive variants are particularly adapted for analytes that havedifficulties in binding two or more affinity counterpartssimultaneously, i.e. relatively small molecules.

Non-Competitive Protocols

These protocols typically utilize non-limiting amounts of one or moreaffinity counterparts to the analyte.

The most important non-competitive protocols are sandwich protocolswhich comprise formation of immobilized or immobilizable complexes inwhich an analyte is sandwiched between two affinity counterparts. One ofthe counterparts is analytically detectable and the other immobilized orimmobilizable and possibly also analytically detectable. The sandwichcomplex is measured in step (iii).

Another non-competitive variant utilizes only one affinity counterpartto the analyte in immobilized or immobilizable form. In this casecomplex formation leads to an immobilized complex, or a soluble complexthat subsequently is immobilized. The immobilized complex as such may bemeasured in step (iii). In one variant the affinity counterpart which isimmobilized or immobilizable has been labeled with an analyticallydetectable group that changes its signal when the analyte binds to theaffinity counterpart.

Non-competitive protocols have their greatest potential for moleculesthat simultaneously can bind two or more affinity counterparts, i.e.large molecules.

Analytically Detectable Reactants.

By the term “analytically detectable” is contemplated that an affinityreactant can be analytically discriminated from other affinity reactantsparticipating in the formation of the complex to be measured in step(iii). Detectability may derive from an inherent property of thereactant, for instance an inherent biological function such as theenzyme activity of an enzyme or Fc-receptor binding activity of variousIg-classes and subclasses, or a separately introduced functionality,e.g. labeling with an analytically detectable tag or label, such asbiotin (=affinity label), enzyme, chromogen, fluorogen, fluorophor,chemiluminscent group, radioactive group etc. Detectability alsoincludes that the formed complex is detectable by itself, for instanceby changing the optic properties of a solution etc.

A detectable label may be combined with a second label selected suchthat the labels together give the appropriate signal when the complex isformed or dissociated. This variant may be illustrated withscintilliation proximity assays (SPA) in which a soluble affinityreactant, which is labeled with tritium, is used together with a solidphase comprising a scintillation substance. When the tritium-labelbecomes incorporated in a complex bound to this kind of solid phase asignal will appear. The principle with interacting labels may also beillustrated with pairs of fluorophores that may be identical ordifferent and with fluorescence-quencher pairs.

An Illustrative Variant of Running Several Samples in Parallel

-   Step (i): The microfluidic device is as discussed above and    comprises k microchannel structures (k is an integer 2 or larger),    each of which comprises an immobilized affinity counterpart to an    analyte or a combination of such counterparts. The affinity    counterparts may or may not differ between the microchannel    structures. The affinity counterparts are preferably located to the    reaction microcavities in which the affinity complex is to be    retained.-   Step (ii): A liquid aliquot (aliquot 1) that contains an unknown    amount of analyte is introduced into n of the k microchannel    structures which contain the affinity counterparts, i.e. aliquot 1¹    into microchannel structure 1, aliquot 1² into microchannel    structure 2 . . . and aliquot 1^(n) into microchannel structure n,    where n is an integer equal or smaller than k. Possibly there is    also introduced an aliquot containing a known amount of analyte into    at least m of the remaining microchannel structures, if any, i.e.    aliquot 1^(n+1) into microchannel structure n+1, aliquot 1^(n+2)    into microchannel structure n+2 . . . and aliquot 1^(n+m) into    microchannel structure n+m, where m is an integer such that n+m is    equal or smaller than k. The aliquots with their possible content of    analyte are then transported through the microchannel structures    such that immobilized complexes will be retained under flow    conditions in the reaction microcavity of each microchannel    structure. For aliquots that contain no analyte there will be no    formation of complex.-   Steps (iii) and (iv): The amount of the immobilized complex in each    reaction microcavity is measured and related to the amount of    analyte in the corresponding starting aliquot/sample.

With respect to reactants and their addition, similar protocols as thosediscussed above may be used for variants B-F below. In these variantsthe demand on using limiting and unlimiting amounts may be lessimportant. For alternatives B-D, the concentration and/or relativeamounts of the affinity reactants used are typically essentiallyconstant for several of the experiments run in parallel according to theinvention. Variants B-F are primarily run under non-diffusion-limitingconditions.

B. Selection of Binders (Analytes) from a Library of Potential BinderCandidates.

Two preferred variants are:

-   A) Different binder candidates and/or combinations of binder    candidates from a library are used in immobilized form in two or    more of the microchannel structures of the microfluidic device    provided in step (i). A soluble form of a known common ligand is    used to form the immobilized complex in step (ii).-   B) The same known immobilized ligand is present in two or more of    the microchannel structures of the microfluidic device provided in    step (i). To each microchannel structure a single binder candidate    or a combination of different binder candidates may be introduced    into each microchannel structure in step (ii).

A larger extent of complex formation in the reaction microcavity of aparticular microchannel structure will suggest that the binder used inthis microchannel structure will have a stronger tendency to form anaffinity complex than a binder giving a lower extent of complexformation in another microchannel structure. This presumes that otherreaction variables are kept essentially constant.

For other variants the meaning of a larger or smaller amount of retainedcomplex in the reaction microcavity will depend on the particularprotocol used. For certain protocols a larger amount will suggest a moreefficient binder compared to a binder resulting in a smaller amount. Forother protocols the opposite will be true.

In both variant (a) and (b) the reaction conditions may or may notdiffer between the individual microchannel structures.

The preferred protocols are the same as for alternative F below.

Formed affinity complexes can be further characterized with respect tothe characteristics of the binder in the complex, for instancestructure, binding characteristics, biological function etc.

C. Determination of Immobilization Techniques and/or Solid Phase thatare Suitable for a Given Affinity Pair.

In this case at least the immobilization technique for the affinityreactant bound to the solid phase and/or the solid phase as such differbetween two or more microchannel structures. The difference may relateto: conditions for physical adsorption, covalent attachment includingkind of covalent bridge, affinity reactants used for affinityimmobilization, kind of solid phase etc.

The meaning of a larger or smaller amount of retained complex in thereaction microcavity will depend on the protocols used. For certainprotocols a larger amount will suggest an immobilization techniqueand/or a solid phase favoring binding compared to a technique givingsmaller amount. For other protocols the opposite will be true.

D. Determination of Suitable Reaction Conditions Related to the Liquid.

In this case at least one reaction variable related to the liquid variesbetween two or more microchannel structures in the microfluidic device.Typical reaction variables are pH, temperature, ionic strength, amountof an inhibitor or a promotor of complex formation, hydrogen-bondbreaking agents, detergents, and flow rate in the microchannelstructures etc.

The meaning of a larger or smaller amount of retained complex willdepend on the protocols used. For certain protocols a larger amount willsuggest an immobilization technique favoring binding compared to atechnique giving smaller amount. For other protocols the opposite willbe true.

E. Determination of a Ligand and/or a Binder with Respect to theirSuitability for Dissociation of their Affinity Complex.

These protocols typically start from microchannel structures in whichthe complex between the ligand and the binder is predispensed orpreformed in immobilized form in the reaction microcavity. The complexmay contain an analytically detectable reactant. Upon dissociation ofthe complex the analytically detectable reactant is released from thecomplex and the remaining amount of complex can be measured directly inthe reaction microcavity or indirectly as the amount of releasedreactant in a separate detection microcavity positioned downstream thereaction microcavity. Alternatively the affinity counterpart that formsthe link to the solid phase comprise a label that change as a functionof the release of the other counterpart.

Suitable reaction variables that may be varied are given under“Definitions” above.

F. Determination of Qualitative Aspects of Complex Formation.

The present invention applies to flow conditions during formation ordissociation of affinity complexes on a solid phase that is retained ina reaction microcavity. One can envisage that the distribution of thecomplex in the solid phase (reaction microcavity) along the flowdirection will reflect affinity constants and rates for association anddissociation etc under the conditions used, and that this distributionwill depend on various factors such as kind of binder and ligand andtheir concentrations, and substitution degree and availability on thesolid phase, kind of solid phase, pH, ionic strength, flow rate etc.

One can therefore conceive that refined information of qualitativeaspects of binding can be accomplished by measuring this distribution,e.g. as the local amount of complex as a function of position in thesolid phase (reaction microcavity) along the flow direction.

The requirement on intra-channel variation in residence time is strict,e.g. the variation should be within mean residence time ±75%, such as±50% or ±25% or ±10% or ±5%.

A simple case is that one starts with a library of compounds (=binders)in which the individual members are of similar size. The library is thentested according to the invention in a microfluidic device in which twoor more of the microchannel structures have the ligand immobilized inthe reaction microcavity. Since the compounds are of the same size, thediffusion will be essentially the same for the members of the library.The local amount of complex as a function of position in the flowdirection of the solid phase will therefore reflect the affinity betweenthe compound and the ligand that has formed the complex in a particularmicrochannel structure. A sharp zone, for instance, in the initial partof the solid phase (reaction microcavity) could suggest a strongeraffinity than a broader more diffuse zone that is located moredownstream.

The distribution of a complex in the solid phase (reaction microcavity)may also be used as a function test for an assay, e.g. of the typediscussed for alternative A above.

This innovative alternative requires that the binding reaction betweenan immobilized ligand and a binder takes place in the reactionmicrocavity and that the complex formed is measured in step (iii). Theimmobilized ligand may be introduced prior to formation of the complex.The ligand and binder may be any of the affinity counterparts givenabove for library members provided the formed complex can be retained insufficient amount in the reaction microcavity. Typically the immobilizedligand is present in an unlimited amount. To facilitate the measurementin step (iii) properly selected detectable reactants can be included inone or more of steps (i)-(iii), e.g. a) the immobilized ligand may havea label that change its signal upon formation of the immobilizedcomplex, b) the binder may have a label meaning that the complex formedin step (ii) will comprise the label, c) a soluble analyticallydetectable affinity counterpart to the binder is used in a sandwichprotocol etc.

A potentially interesting variant includes that two labels that areinteracting with each other when in close proximity are used. A typicalexample is scintillation proximity assays (SPA) in which the ligand islabeled with tritium and the solid phase comprises a scintillationsubstance. SPA has found wide-spread use for screening of drugcandidates, affinity ligands etc with respect to interaction properties.

Other interesting variants include sandwich protocols and displacementprotocols as described above for alternative A with the proviso that thebinder in this variant of the invention replaces the analyte.

A soluble analytically detectable affinity counterpart to the binder ispreferably used when the binder is large and able to bind two affinitycounterparts simultaneously, typically in sandwich protocols. The otherlabeled reactants that are mentioned above have great advantages forsmall binders.

In the context of this aspect of the invention, it may be interesting todetermine relative affinity of a complex, binder and/or ligand inrelation to various members of a library as defined under “Definitions”above.

By adapting the flow rates to implement reactions that are notdiffusion-limited the main feature studied will be the affinityconstants. This in particular applies to affinity reactants that arelarge.

Variants meaning that the formed affinity complex as such is determinedare also possible to use in this alternative (F).

Samples

The samples used in one run of the innovative method correspond to acollection (library) of samples. At least two of the members of thecollection differ with respect to the reaction variable(s) to becharacterized.

Samples that differ with respect to reaction variables that are definedby soluble molecular entities can be introduced into the inlet ports ofthe microchannel structures in step (ii) or in step (iii). In the casethe reaction variables are defined by insoluble entities (solid phases,immobilization techniques etc), the library is preferably provided instep (ii) together with the microfluidic device.

Different protocols may require different kind of samples and viceversa.

For alternatives A and B above, the samples are typically derived frombiological material such as whole blood, plasma, serum, buffy coat,blood cells, semen, cerebrospinal fluid, lymph, lachrymal fluid,ascites, tissue, supernatants from cell lysates, cell culturing etc thathas been treated in various ways in order to give aliquot 1. Includedare also biologically material that has been produced synthetically suchas synthetically produced oligo- and polypeptides, oligo- andpolynucleotides, compounds that potentially are mimicking biologicalmolecules or interactions etc.

For the other alternatives the samples typically are more or lesssynthetic.

Depending on the particular protocol, alternative F may utilize any ofthe abovementioned kinds of samples.

Measuring and Characterization (Step (iii) and Step (iv))

The measurement in step (iii) comprises measuring a) the distribution ofthe affinity complex in the reaction microcavity (solid phase) along theflow direction, or b) the total amount of the complex in the reactionmicrocavity. The latter includes that measurement is carried out only ina certain part of the reaction microcavity as long as this amount isrepresentative for the total amount. Alternative (a) in particularapplies to alternative (F) above.

A number of various ways of measuring immune complexes immobilized to asolid phase by utilizing analytically detectable affinity reactans areknown in the field. In principle most of them can be used in the presentinvention.

Typical examples are spectroscopic methods based on chemiluminescence,bioluminescence, fluorescence with particular emphasis of laser-inducedfluorescence (LIF) etc. As indicated elsewhere in this specificationthese spectroscopic principles often utilize reactants that are labeledwith the corresponding analytically detectable group (label). Otherprinciples are based on signals emanating from the complex as such, forinstance turbidometry.

As already discussed step (iii) may comprise substeps in whichanalytically detectable affinity reactants are introduced forfacilitating the measurement.

The characterization step (step (iv)) means that the measured value instep (iii) is used to characterize an uncharacterized reaction variablein the samples. This is common practice in the field and needs nofurther comments. In some cases when the inventive method results in abinding capacity for an unknown entity characterization for identifyingthe entity may be included in the method aspect of the invention.

The best mode corresponds to the work presented in the experimentalpart.

A Separate Aspect of the Invention.

A separate aspect of the invention is a structural unit (substructure)for reducing inter-channel variations in flow. The unit is illustratedin FIG. 3. The unit comprises (a) an inlet microconduit (302), (b) amicrocavity (304) in which a reaction is to take place under controlledflow conditions, and (c) an outlet microconduit (305). This device ischaracterized in that there are means for creating a pressure drop inthe microcavity (304) and/or in the outlet microconduit (305) downstreamthe microcavity (304) so that the inter-channel variation in residencetime within the microfluidic device for the samples introduced via theinlet microconduits (302) becomes within the limits discussed above.Suitable flow rates through the microcavity are typically within theintervals given above. The means for creating the pressure drop are asoutlined in the earlier part of this specification.

In a preferred variant of this inventive aspect there is also anadditional microconduit (103 in FIGS. 1 a, 306) merging with the inletmicroconduit as illustrated in FIGS. 1 a and b. This additionalmicroconduit may have various functions (vent, inlet etc) and intersectswith the inlet microconduit (302), typically in close proximity to itsjoint with the reaction microcavity (304).

As discussed for FIGS. 1 a and b, and 2 the microchannel structurescomprising this preferred substructure can be present in a substratehaving an axis of symmetry and arranged so that inlet microconduit (302)and the additional conduit (103 in FIG. 1 a) are at a larger radialdistance than the outlet conduit (305) and the reaction microcavity atan intermediary radial distance (304). The substrate may comprise aplurality of this kind of microchannel structures/substructure orientedradially outwards from the axis of symmetry (spinning axis) and arrangedto define two or more annular zones (rings) around the spinning axis, orsectors of such a zone. According to this aspect the microchannelstructures in the same annular zone has the substructure at the sameradial distance while the substructures for other annular zones are at adifferent radial distance. Since it is known that the centrifugal forceat an outer position is larger than at an inner position, outletmicroconduit (105,305) in an outer annular zone shall be longer than inan inner annular zone if the hydrodymanic cross-sectional area is keptconstant and the same liquid flow is desired for all the microchannelstructures of this kind on the substrate.

Thus by varying the design (hydrodynamic cross-sectional area and/orlength) it is possible to place several microchannel structurescomprising the described substructure at different radial distanceswhile maintaining the same flow rate through the reaction microcavity.Inversely this concept makes it possible to construct microfluidicdevices in which there are microchannel structures in which the flowrate through the reaction microcavity will differ in a well-definedmanner.

This aspect of the invention is useful in the method aspect of theinvention (method for the characterization of reaction variables). Thisaspect may also be useful for other microfluidic applications where acontrolled liquid flow is beneficial, e.g. microtitration and chemicalsynthesis in the microformat and other situations where controlledmixing or controlled addition of reagents are needed.

The patent applications given below have been discussed in thisspecification. All of them are hereby incorporated by reference, inparticular those WO applications that designate the US.

WO 9116966, 9615576, WO 9721090, WO 9800709, 9807019, WO 9958245, WO0056808, WO 0146465, WO 0147637, WO 0154810, WO 0185602, U.S. 60/315,471and corresponding International Patent Application, U.S. Ser. No.60/322,622 and the c.i.p. US application filed on Jan. 31, 2002.

EXPERIMENTAL PART

FIGS. 1-2 shows the structure used in this experiment.

A complete myoglobin assay was performed in a disc of the same dimensionas a compact disc (CD). In the disc used there were 24 similarstructures (101,201) in parallel and the structures were divided in 2sets each containing 12 structures (only one set is shown in FIG. 2). Acommon distribution channel (119, 219) connected the 12 structures,through which buffer and reagents were distributed to the 12 reactionstructures via either inlet port (209) or inlet port (210). Everystructure had also its individual inlet unit (108,114) designed with asample receiving structure (111), which has a volume definitionfunction. The dimensions of the channel are indicated in FIGS. 1 a-b.

In order to achieve capillary action in the reaction structures the CDsurface was treated with O₂-plasma, subsequently a plastic lid waslaminated to the CD. According to a standard procedure all structureswere incubated with PEG-PEI WO 0056808 (Gyros AB) and WO 0147637 (GyrosAB). The PEG-PEI molecules bind to the surface and establish a layerwith low protein adsorption. After the PEG-PEI incubation the structureswere thoroughly washed with water.

The immunoassay was performed in an automated system. The system(instrument prototype, Gyros AB, Uppsala, Sweden) was equipped withCD-spinner, holder for Microtiter plates (MTP) and a robotic arm with aholder for a capillary connected to a syringe pump and 8 slit pins. Thecapillary transferred all reagents and buffers from a MTP to either ofthe two inlet units (209,210) of the common distribution channel (119)in the disc. The slit pins transferred individual samples from a MTP tothe individual sample inlet units (108) in the disc.

The instrument prototype is a fully automated robotic system controlledby application-specific software. Microplates containing samples orreagents are stored in a carousel within the system. A high precisionrobot transfers samples from microplates or containers into themicroworld of the CD. CDs are moved to the spinning station for theaddition of samples and reagents. An application-specific method withinthe software controls the spinning at precisely controlled speedscontrols the movement of liquids through the microstructures as theapplication proceeds.

Polystyrene particles (15 μm, Dynal Particles AS, Norway) were selectedfor the solid phase. The beads was modified by passive adsorption ofphenyl-dextran to create a hydrophilic surface and were subsequentlycovalently coupled with a monoclonal antimyoglobin 8E.11.1 (LabAs,Tartu, Estonia) using CDAP chemistry (Kohn & Wilchek, Biochem. BiophysRes. Comm. 107 (1982) 878-884).

After coupling with the antibody, a suspension of the particles wasdistributed in the common distribution channel (119) via inlet unit(109) and moved through the structure by centrifugal force. Thecentrifugal force combined with the vents (122) divide the suspension inthe channel in equal portions each of which forms a bed of packedparticles (column) in the reaction microcavity (104) against the dualdepth in front of the outlet conduit (105). The approximate volume ofthe column was 10 nl. The columns were washed once with 0.01 MPhosphate-buffer, pH 7.5 containing 0.15 M NaCl via the commondistribution channel (119). Every addition of solution delivers 200 nlliquid to the individual column. One addition of washing solution is 20times the column volume, which ensures a very efficient washing

To demonstrate the myoglobin assay in the system a 6-point standardcurve was created. The myoglobin samples (diluted in PBS, 1% BSA) withconcentrations in the range of 0-1620 ng/ml where distributed to theindividual inlet units by slit pins. The sample volume 100 nl wasdefined in the sample receiving structure (111) (during the first twostep in the spin flow method). To reach favourable kinetic conditionunder the capturing step (for myoglobin to bind) the flow rate of thesample should not exceed 1 nl/sec. The sample flow rate was controlledby the spin velocity (spin fowl). After sample capturing the columnswere washed twice by addition of PBS, 0.01% Tween to the commondistribution channel followed by a spin step. Detection antibodies inexcess were applied next via the common distribution channel, these weremonoclonal antimyoglobin 2F9.1 (LabAs,Tartu) and a similar slow flowrate (spin flow 2) was used. The detecting antibodies were labelled witha fluorophor Alexa 633 (Molecular Probes, Eugene, USA). Excess oflabelled antibody was washed away in 4 additions of PBS-T.

The completed assay was analysed in a laser induced fluorescence (LIF)detector. The detector moved radially along the column while the discwas rotating. The results are presented in FIG. 4. Signal intensity wastypically concentrated to the top of the column.

The design of the Laser Induced Fluorescence (LIF) module wasconstructed for quantitative measurement of fluorescence in themicrofluidic device (disc of CD format) used in the experiments.

The disc was rotated by means of a motor on a rotatable shaft; the speedcould be varied from 60 to 3000 rpm. By means of an encoder and a fixedhome position on the CD the position of each structure of interest couldbe stored in a file.

A pick-up head was constructed. It consisted of a laser whose beam wasreflected on a dichroic mirror and focused onto the structure ofinterest in the disc through a 5× objective. The epi-fluorescent lightpassed through the dichroic mirror and through a band-pass filter,optimised for the flourochrome at hand. It was focused onto a PMT bymeans of an aspheric lens.

By rotating the disc while measuring a signal across the structure atrace was obtained corresponding to the fluorescence in the structure.By moving the pick-up means of a motor in radial direction whilerotating the disc a measurement of the total fluorescence in thestructure was obtained.

The detector is described in U.S. Ser. No. 60/322,622, SE 0103118-6, SE010446-9 and US application filed on Jan. 31, 2002 (“Arrangement andmethod for measurement in spinning microfluidic devices”).

An overview of the run method in the system is presented in Tab 2

TABLE 1 An overview of the run method performed in the system Run methodSpin profile Wash of CD structures Spin1 5700 rpm 15 s Transfer of beadsSpin2 4500 rpm 10 s, 5000 rpm 10 s Wash of beads Spin3 5800 rpm 15 s,6700 rpm 5 s Transfer of samples Spin flow1 1200 rpm 2 s, 2200 rpm 0.5s, from 1700-2500 rpm in 90 sec. 3000 rpm 15 s, 5500 rpm 5 s Myoglobinwash 1 Spin4 4500 rpm 20 s, 5700 rpm 5 s Myoglobin wash 2 Spin5 4500 rpm20 s, 5700 rpm 5 s Transfer of conjugate Spin flow 2 2500 rpm 0.6 s,1500-2500 rpm in 120 sec. 3000 rpm 15 s, 5500 rpm 5 s Conjugate wash1Spin6 4500 rpm 20 s, 5700 rpm 5 s Conjugate wash2 Spin7 4500 rpm 20 s,5700 rpm 5 s Conjugate wash3 Spin8 4500 rpm 20 s, 5700 rpm 5 s Conjugatewash4 Spin9 4500 rpm 20 s, 5700 rpm 5 s Detection

Certain innovative aspects of the invention is defined in more detail inthe appending claims. Although the present invention and its advantageshave been described in detail, it should be understood that variouschanges, substitutions and alterations can be made herein withoutdeparting from the spirit and scope of the invention as defined by theappended claims. Moreover, the scope of the present application is notintended to be limited to the particular embodiments of the process,machine, manufacture, composition of matter, means, methods and stepsdescribed in the specification. As one of ordinary skill in the art willreadily appreciate from the disclosure of the present invention,processes, machines, manufacture, compositions of matter, means,methods, or steps, presently existing or later to be developed thatperform substantially the same function or achieve substantially thesame result as the corresponding embodiments described herein may beutilized according to the present invention. Accordingly, the appendedclaims are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

1. A microscale method for the characterization of at least one reactionvariable that influences the dissociation of an affinity complexcomprising a ligand and a binder, which have affinity for each other,said method comprising the steps of: (i) providing a microfluidic devicecomprising a plurality of microchannel structures that are under commonflow control, each microchannel structure comprising a reactionmicrocavity; (ii) performing essentially in parallel an experiment ineach of two or more of the plurality of microchannel structures, theexperiment in these two or more microchannel structures comprisingdissociating under flow conditions the immobilized form of the complexwhich has been introduced prior to step (ii), said at least one reactionvariable being different or uncharacterized for said two or moremicrochannel structures; (iii) measuring the presentation of the complexin said reaction microcavity in each of said two or more microchannelstructures; and (iv) characterizing said at least one reaction variablebased on the values for presentation obtained din step (iii).
 2. Themethod of claim 1, wherein each of the microcavities of said two or moremicrochannel structures comprises a solid phase to which an affinityreactant which is capable of being incorporated into the affinitycomplex retained in step (ii) is attached.
 3. The method of claim 1,wherein (a) the microfluidic device comprises a substrate having an axisof symmetry, (b) each microchannel structure is oriented relative theaxis of symmetry with an inlet port at shorter radial distance than thereaction microcavity, and (c) the substrate is spun around its axis ofsymmetry to drive liquid within the microchannel structures.
 4. Themethod of claim 1, wherein a) the microfluidic device comprises asubstrate having an axis of symmetry, b) each microchannel structure isoriented radially relative the axis of symmetry with the reactionmicrocavity at a larger radial distance than a substructure deliveringliquid to the reaction microcavity, and c) the substrate is spun aroundits axis of symmetry to drive liquid within the microchannel structures.5. The method of claim 1, wherein each of the microchannel structurescomprises a flow restriction downstream the reaction microcavity, whichcreates a pressure drop that restricts the flow through the reactionmicrocavity.
 6. The method of claim 1, wherein step (iii) is performedby measuring (a) distribution of the complex in the reaction microcavityalong the flow direction, or (b) the total amount of the complex in thereaction microcavity.
 7. The method of claim 1, wherein step (iii)comprises measuring the total amount of the complex in the reactionmicrocavity.
 8. The method of claim 1, wherein each of said experimentscomprises formation of an immobilized form of the complex within thereaction microcavity.
 9. The method of claim 1, wherein said experimentcomprises dissociating under flow conditions an immobilized form of thecomplex which complex is included in the microfluidic device provided instep (i).
 10. The method of claim 1, wherein said at least one reactionvariables that varies between the microchannel structure is selectedamongst group 1 or group 2 reaction variables.
 11. The method of claim1, wherein the formation of the complex that is retained in the reactionmicrocavity is part of a competitive/inhibition protocol or anon-competitive protocol.
 12. The method of claim 11, wherein said atleast one reaction variable is related to an affinity reactant.
 13. Themethod of claim 12, wherein said at least one reaction variable isrelated to the amount of said affinity reactant.
 14. The method of claim12, wherein said at least one reaction variable is related to affinityof an affinity reactant.
 15. The method of claim 1, wherein step (iii)comprises determining the distribution of the complex along the flowdirection in the reaction microcavity in each of said two or moremicrochannel structures.
 16. The method of claim 15, wherein step (iv)comprises that the relative affinity for the ligand and binder as afunction of the variation in said at least one reaction variable ischaracterized.